Autoradiography is the production of an image in a photographic emulsion by a relatively labelled substance. Autoradiography is an important method in biological, biochemical and clinical investigation and analyses. The isotopes most often used with this technique are 3H, 14C, 35S, 125I, and 32P. The low energy of the disintegration particles of 3H, 14C and 35S prevents the efficient exposure of the X-ray film used to detect this radioactivity. The researcher must use large amounts of radioactivity or very long film exposure times.
To overcome the problems of sensitivity and long exposure times, researchers incorporate scintillators (or fluors) into their separation media. After the mixtures of radioactively labelled samples are separated by conventional separation techniques, the fluors are applied to the media. The radiation energy emitted by the unstable isotopes is absorbed by the fluors present in the system and these fluors emit light at a specific wavelength. The light given off by the fluors exposes the film much more efficiently than the radiation energy itself. This detection method is commonly known as fluorography.
Fluorography is useful with a wide variety of separation media. For example, in chromatography and electrophoresis, the radioactive material to be detected is absorbed or adsorbed according to conventional techniques in or on an organic or inorganic absorbent or adsorbent layer or column of separation medium or material, e.g. silica gel, alumina, cellulose, polyamide, polyacrylamide, cross-linked dextran, agarose, nylon, nitrocellulose, etc., which is usually supported on a plate, e.g. glass or plastic sheet.
In spite of its importance, fluorography is not an easy method to apply. In the case of thin layer chromatography, the fluor is dissolved in a suitable carrier solvent and then sprayed onto the thin layer separation medium, e.g., a paper strip containing a radioactive sample. After the carrier solvent has been evaporated, the chromatogram is placed in direct contact with the photographic emulsion. This sandwich is left undisturbed for a sufficient time to achieve exposure. This method works because the layer is so thin that the radioactive emitters are close enough to the surface to interact with a surface layer of fluor. However, in such a system, it is difficult to evenly distribute the fluor, the radioactive material may be spread and diffused by the carrier solvent, and the small crystals of fluor tend to be so loosely bound that great care must be exercised in handling the sample.
Further, it is often desirable to employ a separation medium which is thicker, i.e. greater than about 0.1 mm. In this case, the technique of coating the surface by spraying is no longer adequate. The isotopes used in these techniques have a relatively low energy and, therefore, the radiation has short path length. It is, therefore, necessary for the fluor to be in close proximity to the radioactive label such that the radiation energy will interact with the fluor and thus be amplified. In relatively thick layers, it is necessary to uniformly impregnate the layer with fluors. If the separation media is coated on the surface only, the vast majority of the disintegration particles will be absorbed before they reach the fluors or the film and go undetected. Accordingly, it is necessary to somehow uniformly transport the fluor into this interior of the separation medium. One method for accomplishing this transportation is by soaking the separation medium of absorbent or adsorbent material in a bath containing the fluor dissolved in a suitable carrier.
Many methods for incorporating fluors into separation media appear in the literature. Three methods used for thin layer chromatography are described in Bonner and Stedman, Anal. Biochem., Vol. 89, 247-256, (1978) incorporated herein by reference.
The first method uses 2-methylnaphthalene, which is described as being a scintillation solvent for use in solid systems by analogy to scintillation fluids, which many times contains a solvent in addition to the scintillator. As in liquid systems, the solvent molecules collect the energy from the emitted beta radiation and transfer it to 2,5-diphenyloxazole molecules, which emit photons of light at the proper wavelength to expose the X-ray film. This method comprises dipping the dried, thin-layer plates in a solution of 2-methylnaphthalene which has been melted and which contains 0.4% (w/v) of 2,5-diphenyloxazole until they are soaked and then removing the plates from the solution. When the solution cools and solidifies, the plate is placed against film and exposed. An alternative, if spraying is more desirable, is to replace 10% of the 2-methylnaphthalene with toluene to make the solution a liquid at room temperature.
The second method involves dipping the plates in an ether solution containing between 7% and 30% (w/v) of 2,5-diphenyloxazole, drying the plates and exposing to film, with better sensitivity being seen as the 2,5-diphenyloxazole (or PPO) concentration increases.
The third method involves dipping the thin layer plate in melted PPO until soaked, removing and then heating until the excess PPO has drained off, and exposing to film as above.
These methods are helpful for thin layer chromatography, but have serious drawbacks when applied to gel electrophoresis. The gels used generally contain &gt;80% water and more often &gt;90% water. The fluors and solvents used in these methods are not water soluble or water miscible. This will prevent efficient and uniform transport of the fluors to the interior of the gel. The fluors tend to precipitate on the surface of the gel. The suggested organic solvents cause drastic shrinkage of the gel, prevent impregnation, and lead to distortion of the gel.
One method for increasing the absorption ability of 2,5-diphenyloxazole is to use scintillation solvents. The solvent molecules which are more efficient absorbers of energy from the emitted beta radiation convert the energy to photons of light, and transfer it to 2,5-diphenyloxazole molecules which then emit photons of light detected by X-ray films. This method is described by Bonner and Stedman in Anal. Biochem. Vol. 89, pages 247-256, (1978), for thin layer chromatography. One problem is that neither 2,5-diphenyloxazole nor 2-methylnaphthalene, the combination of fluors described by Bonner and Stedman, is soluble nor miscible in water to any appreciable extent. Accordingly aqueous polyacrylamide or agarose gels are not impregnated with 2,5-diphenyloxazole or 2-methyl naphthalene while in the hydrated state.
Dennis C. Fost, U.S. Pat. No. 4,293,436, Oct. 6, 1981 described a highly sensitive and efficient autofluorographic technique for weak radioactive emitters by impregnating the aqueous separation medium, including electrophoresis gels, with a water-soluble or water-miscible lower alkyl carboxylic acid and alcohol combination in which a scintillator fluor has been dissolved, followed by precipitation of the fluor within the gel by aqueous soaking. However, this procedure suffers from disadvantages. The organic solvents of the impregnation step shrink the gel. The water used in the precipitation step then causes the gel to expand. This distortion can adversely affect the resolution of the image produced.
Another fluorographic system has been described in U.S. Pat. No. 4,522,742, issued June 11, 1985 to Lee, Feierberg and O'Brien. In that method, the aqueous separation medium to be subjected to fluorography is impregnated in an aqueous autoflourographic enhancer containing water soluble fluors which eliminate the problems associated with the impregnation of gels, and permit wider and more convenient use of fluorography. This water-based system does not, however, work effectively in very thin gels (&lt;1.0 mm) or gels with &lt;5% acrylamide or agarose. Unlike the systems based on organic solvent impregnation and water precipitation, this aqueous system transports the fluors into the gel without exchanging the solvent and without precipitating the fluors. Prior to film exposure, the gels must be dried. The most common technique involves drying the gel onto a piece of filter paper with heat and vacuum. As soon as the vacuum is applied, the majority of the water is suctioned out of the gel rather than evaporated; and some of the water soluble fluor is pulled out with the water. For gels with a small mass, enough fluor is pulled out to seriously decrease the enhancement of the film exposure.
It is the purpose of the invention to create a new system where the fluors are precipitated within the gel using only aqueous solutions, thus having the advantages of precipitated fluors without the disadvantages of hazardous organic solvents.